Treponema pallidum belongs to the bacterial family of spirochetes and is the causative agent of syphilis. Syphilis, also called Lues, is mainly transmitted by sexual contact. Treponema pallidum can also pass from mother to baby during pregnancy. The disease is characterized by distinct clinical stages and long periods of latent, asymptomatic infection. Many people do not notice symptoms and thus are unaware of their syphilis infection for years. The primary infection is confined and usually causes a small painless ulcer (primary stage, “Lues I”). If left untreated by penicillin, the disease proceeds to the secondary stage Lues II (about eight weeks after infection), which entails flu-like symptoms, non-itchy skin rash and swollen lymph nodes. After some years, at stage Lues III, syphilitic nodes appear throughout the body. The final stage (Lues IV) is characterized by destruction of the central nervous system eventually leading to neurological and cardiological disorders, general paralysis, ataxia, dementia and blindness.
Although effective therapies have been available since the introduction of penicillin in the mid-20th century, syphilis still remains an important global health problem. It is mandatory to identify patients with Treponema infection, to support antibiotic therapy and thus to prevent spread of syphilis. As a consequence, it is necessary to provide reliable diagnostic tools such as immunoassays for the detection of antibodies against Treponema pallidum. Yet, in order to be used as specific compounds in serological applications, recombinant proteins have to meet several requirements such as solubility, stability and antigenicity.
One of the membrane-associated polypeptides of Treponema pallidum (the causative agent of Syphilis infections) is TpN47, a large protein that consists of 434 amino acid residues. TpN47 has been ascribed immunodominance in the humoral immune response against Treponema (N Rostopira et al., Folia Microbiol. (2003) 48 (4), 549-553), and antibodies towards TpN47 are frequently found in human sera from Treponema-infected individuals. Thus, a soluble and antigenic variant of recombinant TpN47 would be an invaluable ingredient to establish an immunoassay for detection of Treponema antibodies that combines high sensitivity and specificity. In the best case, such an antigen should enable the detection of both IgG and IgM molecules.
Recombinant variants of TpN47 have been described in literature. In the Journal of Immunology (1996) July 15; 157(2):720-31, Baughn et al. report on an epitope scan of 12-mer peptides (overlap 8 amino acid residues, offset 4 amino acid residues) encompassing the entire sequence of TpN47. Based on this scan, the authors describe as many as ten immunodominant TpN47 fragments, ranging from 9 to 29 amino acid residues in length. The crystal structure and the domain topology of TpN47 has also been described (Journal of Biological Chemistry (2002), 277 (4), 41857-41864, Deka et al.). Immunoassays for detecting Treponema antibodies are known in the art. For example, Rostopira et al. (Folia Microbiol. 48(4), 549-553, 2003) describe an immunoassay for diagnosing syphilis using a combination of TpN17 and TpN47 antigens, identifying TpN47 as one of the immunodominant antigens. In this publication full-length TpN47 was used as an antigen.
We overproduced a full-length variant of TpN47 in an E. coli host (BL21/DE3) and succeeded in preparing the TpN47 antigen to homogeneity. Yet, our initial experiments with the full-length version of TpN47 unambiguously revealed that this protein tends to aggregate at temperatures above 35° C. Despite the fusion of tandem chaperone fusions such as EcSlyD-EcSlyD or even (the more thermostable) EcSlpA-EcSlpA, full-length TpN47 inevitably aggregated into a high-molecular-weight associate at temperatures above 35° C. Since Treponema pallidum is known as a rather temperature-sensitive pathogen, the finding that one of its major membrane proteins shares temperature sensitivity may not seem too surprising. Anyway, thermally induced aggregation processes of proteinaceous ingredients in immunoassays usually result either in a loss of signal or in a loss of specificity. Thus, the fact that full-length TpN47 (even in fusion with solubility-enhancing chaperones such as SlyD or SlpA) tends to aggregate even at moderately elevated temperatures (>35° C.), clearly precludes this molecule from simple and straightforward applications in a sensitive immunoassay of the DAGS format.
Despite the detailed structural knowledge on TpN47 (Deka et al. Journal of Biological Chemistry (2002), 277 (4), 41857-41864), the prior art does neither disclose the pronounced thermolability of TpN47 nor does it disclose TpN47 antigen variants that overcome the problem of thermally induced aggregation and the concomitant loss of sensitivity in immunoassays aiming at the detection of antibodies against Treponema pallidum in a sample.
Guo et al. (Xiamen Daxue Xuebao-Ziran Kexue Ban (2008), 47(6), 874-878) describe specific soluble TpN47 N- or C-terminal truncated mutants that are recombinantly expressed in E. coli. However, the problem of thermal instability and tendency to aggregation of full-length TpN47 protein is not addressed. In addition, the data of Guo et al. suggest that a combination of the domains C and D (C190) of TpN47 almost equals the antigenicity of the full-length TpN47 protein.
The stability problem has been solved in the current invention by generating soluble, stable and immunoreactive variants of Treponema pallidum antigen 47 (TpN47 antigen) comprising at least domain B (aa 63-174) of the TpN47 protein molecule with the proviso that the antigens lack domain C (aa 224-351) of TpN47, wherein the TpN47 antigen is fused with a chaperone.
A further solution of this problem is a soluble TpN47 antigen comprising domains B and D (aa 418 to 529 and 707 to 789 of SEQ ID NO. 1) or a TpN47 antigen comprising domains A+B and D (aa 381 to 578 and 707 to 789 of SEQ ID NO. 1). Both variants lack domain C of TpN47.